European Food Research and Technology (2024) 250:2255–2272

https://doi.org/10.1007/s00217-024-04535-7

ORIGINAL PAPER

Detection of honey adulteration by characterization

of the physico‑chemical properties of honey adulterated
with the addition of glucose–fructose and maltose corn syrups
Ramazan Gün1,2 · Mehmet Murat Karaoğlu3

Received: 29 December 2023 / Revised: 12 March 2024 / Accepted: 16 March 2024 / Published online: 21 April 2024
© The Author(s) 2024

Abstract
This study aimed to experimentally investigate the effect of sugar syrup additions on quality measurements of honey and
to detect adulteration. For that purpose, two different pure blossom honey samples were adulterated by directly mixing 0%,
5%, 10%, 20%, 30%, 40%, and 50% of commercially available glucose–fructose corn syrup and maltose corn syrup. In this
regard, key physico-chemical properties like moisture, pH, free acidity, proline, diastase number, color (L, a, b and Delta-E),
electrical conductivity, HMF, sugar profile (glucose, fructose, sucrose and maltose), and C4 sugar analysis were tested. The
results of the individual analysis of moisture, pH, free acidity, proline, diastase number, color, electrical conductivity, and
HMF failed to detect sugar syrup adulteration. However, when principal component analysis (PCA) was utilized to analyze
the data gathered from these tests, adulterations at all-syrup ratios (5–50%) were successfully detected.

Extended author information available on the last page of the article

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2256 European Food Research and Technology (2024) 250:2255–2272

Graphical Abstract

Keywords Adulteration · Physico-chemical properties · Pure honey · Sugar syrups

Introduction high population: 3.38 g/day and 0.62 g/day in Turkey and
worldwide, respectively.
Honey is a natural sweet food produced by honey bees It can be said that adulteration of honey is inevitable since
from different sources, such as plant nectar, secretions of the amount of honey production is very low and not enough
plants, and excretion of plant-sucking insects [1]. Honey when the world population is taken into consideration.
is known as the oldest natural sweetener since ancient As a natural food product, honey is counted as one of the
times, and its consumption and popularity have been grow- major sources of income and well-being for many people in
ing for centuries due to its therapeutic properties, high the food sector. Honey adulterations are commonly made by
nutritional values as well as its uniquely pleasant aroma beekeepers and sellers in two different approaches which are
and sweetness. Although demand for authentic honey by directly adding sugar syrups and indirectly feeding honey-
consumers has been growing all around the world over bees with sugar syrups. Low-cost commercially available
the last years, the production and productivity of natu- syrups can be listed as high-fructose corn syrup (HFCS),
ral honey have been gradually decreased from 1.882.479 inverted syrup, sugar cane syrup, sugar beet syrup, corn
tons to 1.770.119 tons and from 20.5 kg/hive to 18.83 kg/ syrup, sucrose syrup inulin syrup, date syrup, and agave
hive, respectively, between 2017 and 2020 on a global syrup [4–6].
scale [2]. As illustrated in Table 1, although Turkey is the Adulteration in honey can lead to customer mispercep-
world’s second-largest honey producer with a production tions and make consumers think that authentic honey is
of 104.077 tons, consumption of honey is very low due to expensively sold by producers. Furthermore, adulterated
foods adversely affect public health as substances added
European Food Research and Technology (2024) 250:2255–2272 2257

Table 1  Honey production statistics In this study, key physico-chemical tests have been
Parameters Turkey Worldwide Source carried out to discriminate pure honeys from adulterated
honeys.
Number of beehive 8.179.085 93.999.656 [2]
Honey production (tons) 104.077 1.770.119 [2]
Honey yield (kg/hive) 12.72 18.83 [2] Materials and methods
Population 84.339.000 7.794.799.000 [3]
Calculated consumption of 3.38 0.62 – Materials
honey (g/capita/day)

Sample collection and preparation

Pure blossom honey samples were directly collected on

Table 2  The criteria of the physico-chemical properties of blossom
honey [10] August 15–16, 2020 from beekeepers situated in Bingöl
province, Turkey. In the laboratory, a 0.5 mm sieve (Retsch
Parameters Permitted maximum ASTM E11) was used to remove impurities from the honey
and minimum levels
samples. Adulteration agents, glucose–fructose corn starch
Moisture (max.) 20% syrup (SRF 30 Hüner), and maltose corn starch syrup (SM
Free acidity (max.) 50 mEq/kg 40 Hüner) were purchased from Erseval candy factory
Electrical conductivity (max.) 800 μS/cm located in Erzurum province, Turkey.
Proline (min.) 300 mg/kg
Diastase number (min.) 8 (DN)
Sucrose (max.) 5 (g/100 g) Sample production and design process
Maltose (max.) 4 (g/100 g)
Fructose + glucose (min.) 45 (g/100 g) The pure honey samples were adulterated with the syrups
Fructose/glucose 0.9–1.4 at different levels (0%, 5%, 10%, 20%, 30%, 40% and 50%
δ13Cbal value − 23 and more negative w/w). To prepare homogeneous adulterated samples, the
δ13Cprotein—δ13Cbal value − 1 or more negative mixtures were left in a controlled water bath at 35 ℃ for
C4 sugar ratio obtained from δ13Cprotein and %7 1 h. Then these were stirred vigorously with a glass rod
δ13Cbal (max.) for 3 min. Finally, prepared samples were stored at room
HMF (max.) 40 mg/kg temperature until use. A total of 28 samples were designed
including 24 honey–syrup samples, two pure honeys, and
two syrup samples. In the sample ID column of the tables
to them may cause allergic and toxic reactions in human (see Tables 3, 4, and 5), the H1 and H2 represent pure hon-
bodies. By providing an artificial market advantage with eys, GFS and MS stand for glucose–fructose and maltose
adulterated foods, especially bee and bee products produc- syrups, respectively, and the following numbers refer to the
ers, unfair competition in the food industry will arise and ratio of the syrups. For instance, sample ID of H1GFS50
current stability will result in deterioration [7, 8]. Adul- means H1 honey involving GFS syrup with a ratio of 50%.
teration of honey made by sugar syrups has a negative
impact on the chemical and physical properties of honey Methods
such as the enzymatic activity, electrical conductivity,
HMF content, sugar profile and specific compound con- Moisture content, pH, free acidity, electrical conductivity,
tents, etc. According to most national and international and proline content analyses were determined according to
institutions and organizations, including the Turkish Food harmonized methods of the International Honey Commis-
Codex, Council Directive Codex Alimentarius, FAO, and sion (IHC) [36].
WHO, adulteration in honey is prohibited. No food ingre-
dients or substances can be added to honey and honey
can only be blended. Aims of these communiqués are to Moisture content
produce honey hygienically and preserve the unique prop-
erties of honey during the production processes (produc- The moisture content value was determined from the refrac-
tion, preparation, processing, storage, and transportation) tive index of the sample at 20 ℃ using an Abbe refractom-
until it reaches the final consumer [9, 10]. The criteria of eter. The obtained refractive index was converted into mois-
the physico-chemical properties of blossom honey set by ture content (g/100 g) using the standard table.
Turkish Honey Communiqué are given in Table 2 below.
 2258

Table 3  Moisture, pH, free acidity, diastase number, electrical conductivity, HMF, proline, and color parameters of the honey samples
Sample ID Ratio % Moisture (%) pH Free acidity Diastase num- Electrical HMF (mg/kg) Proline (mg/ Color Honey quality
(mEq/kg) ber (DN) conductivity kg)
(μS/cm) L a b ΔE

H1 0 14.96 ± 0.00 3.70 ± 0.02 27.00 ± 0.50 28.93 ± 2.01 307.00 ± 1.00 7.12 ± 0.00 965.54 ± 5.74 75.05 ± 0.04 3.59 ± 0.58 49.36 ± 0.07 0.00 Pure
H1GFS5 5 15.03 ± 0.12 3.73 ± 0.00 24.33 ± 0.29 28.93 ± 2.01 295.00 ± 1.00 7.97 ± 0.13 902.53 ± 2.60 75.55 ± 0.04 3.33 ± 0.02 47.47 ± 0.24 2.03 ± 0.26 Pure
H1GFS10 10 15.09 ± 0.12 3.73 ± 0.00 23.67 ± 0.29 27.60 ± 0.29 283.67 ± 0.58 8.36 ± 0.22 830.96 ± 6.02 76.54 ± 0.15 2.93 ± 0.01 45.95 ± 0.84 3.82 ± 0.74 Pure
H1GFS20 20 15.36 ± 0.00 3.74 ± 0.02 20.83 ± 0.29 26.85 ± 1.60 256.67 ± 0.58 9.08 ± 0.32 703.76 ± 6.50 79.27 ± 0.22 1.97 ± 0.02 43.51 ± 0.09 7.41 ± 0.23 Pure
H1GFS30 30 15.56 ± 0.00 3.77 ± 0.01 19.17 ± 0.29 25.00 ± 0.00 234.00 ± 1.00 9.95 ± 0.42 656.11 ± 14.04 81.15 ± 0.28 1.13 ± 0.02 39.67 ± 0.05 11.72 ± 0.21 Pure
H1GFS40 40 15.89 ± 0.12 3.77 ± 0.01 16.33 ± 0.58 23.61 ± 2.41 210.67 ± 0.58 10.61 ± 0.30 513.38 ± 11.60 83.25 ± 0.29 0.34 ± 0.02 36.17 ± 0.10 15.87 ± 0.18 Pure
H1GFS50 50 15.96 ± 0.00 3.76 ± 0.02 14.00 ± 0.00 17.89 ± 1.07 185.00 ± 0.46 11.94 ± 0.08 438.22 ± 7.92 85.08 ± 0.27 -1.00 ± 0.13 31.34 ± 0.03 21.00 ± 0.27 Pure
GFS 100 17.16 ± 0.00 5.19 ± 0.07 1.63 ± 0.11 0.00 ± 0.00 5.20 ± 0.10 16.96 ± 0.27 53.14 ± 0.47 95.39 ± 0.15 -1.50 ± 0.09 1.08 ± 0.02 52.53 ± 0.15 –
H1 0 14.96 ± 0.00 3.70 ± 0.02 27.00 ± 0.50 28.93 ± 2.01 307.00 ± 1.00 7.12 ± 0.00 965.54 ± 5.74 75.05 ± 0.04 3.59 ± 0.58 49.36 ± 0.07 0.00 Pure
H1MS5 5 14.89 ± 0.12 3.70 ± 0.00 24.00 ± 0.50 28.93 ± 2.01 295.33 ± 1.15 7.39 ± 0.13 916.40 ± 6.41 76.04 ± 0.03 3.44 ± 0.02 47.92 ± 0.06 1.81 ± 0.06 Pure
H1MS10 10 14.76 ± 0.00 3.71 ± 0.01 23.50 ± 0.50 28.93 ± 2.01 284.00 ± 0.00 7.42 ± 0.08 858.18 ± 6.48 76.61 ± 0.34 3.01 ± 0.01 46.35 ± 0.02 3.47 ± 0.30 Pure
H1MS20 20 14.49 ± 0.23 3.72 ± 0.01 20.50 ± 0.76 27.77 ± 0.00 257.33 ± 1.53 7.55 ± 0.06 746.69 ± 11.21 77.58 ± 0.08 2.06 ± 0.00 42.45 ± 0.11 7.53 ± 0.17 Pure
H1MS30 30 14.36 ± 0.00 3.72 ± 0.00 18.67 ± 0.29 26.85 ± 1.60 233.00 ± 1.00 7.64 ± 0.11 668.18 ± 28.68 80.39 ± 0.06 1.12 ± 0.01 38.87 ± 0.01 12.04 ± 0.17 Pure
H1MS40 40 14.23 ± 0.12 3.73 ± 0.01 16.17 ± 0.29 23.61 ± 2.41 206.00 ± 0.00 7.84 ± 0.03 540.00 ± 15.99 82.15 ± 0.05 0.39 ± 0.01 34.91 ± 0.06 16.42 ± 0.20 Pure
H1MS50 50 14.09 ± 0.12 3.73 ± 0.01 13.83 ± 0.29 18.51 ± 0.00 181.73 ± 0.15 8.21 ± 0.02 486.24 ± 4.12 83.35 ± 0.36 -0.80 ± 0.04 29.92 ± 0.38 21.49 ± 0.39 Pure
MS 100 13.76 ± 0.00 5.42 ± 0.00 1.40 ± 0.10 0.00 ± 0.00 4.10 ± 0.00 10.53 ± 0.15 85.88 ± 0.39 95.31 ± 0.27 -1.40 ± 0.04 0.75 ± 0.04 52.80 ± 0.22 –
H2 0 14.56 ± 0.00 3.76 ± 0.03 18.50 ± 0.00 32.74 ± 2.57 242.00 ± 1.00 5.31 ± 0.06 587.37 ± 13.23 83.35 ± 0.32 -0.73 ± 0.01 29.11 ± 0.03 0.00 Pure
H2GFS5 5 14.63 ± 0.12 3.75 ± 0.01 18.00 ± 0.00 31.25 ± 0.00 233.00 ± 1.00 5.86 ± 0.05 537.13 ± 18.49 84.09 ± 0.11 -0.77 ± 0.01 28.49 ± 0.03 0.97 ± 0.16 Pure
HSGFS10 10 14.69 ± 0.12 3.76 ± 0.01 17.16 ± 0.29 31.25 ± 0.00 224.00 ± 1.00 6.47 ± 0.16 502.52 ± 2.51 85.14 ± 0.94 -0.86 ± 0.02 27.26 ± 0.36 2.69 ± 0.85 Pure
H2GFS20 20 14.96 ± 0.00 3.76 ± 0.01 15.83 ± 0.29 28.93 ± 2.01 206.00 ± 1.00 7.59 ± 0.08 470.98 ± 9.10 85.94 ± 0.30 -1.04 ± 0.02 25.15 ± 0.05 4.75 ± 0.24 Pure
HSGFS30 30 15.16 ± 0.00 3.75 ± 0.01 14.17 ± 0.29 25.92 ± 1.60 188.10 ± 0.36 8.63 ± 0.04 412.87 ± 12.15 87.64 ± 0.05 -1.25 ± 0.01 22.75 ± 0.22 7.69 ± 0.29 Pure
HSGFS40 40 15.63 ± 0.12 3.76 ± 0.01 12.50 ± 0.50 23.61 ± 2.41 168.57 ± 0.25 9.79 ± 0.06 359.29 ± 4.70 88.40 ± 0.20 -1.34 ± 0.02 20.04 ± 0.11 10.40 ± 0.02 Pure
HSGFS50 50 15.83 ± 0.12 3.79 ± 0.02 11.50 ± 0.00 20.06 ± 1.34 151.20 ± 0.26 10.70 ± 0.09 314.96 ± 11.72 89.78 ± 0.41 -1.36 ± 0.02 17.02 ± 0.17 13.72 ± 0.22 Pure
GFS 100 17.16 ± 0.00 5.19 ± 0.07 1.63 ± 0.11 0.00 ± 0.00 5.20 ± 0.10 16.96 ± 0.27 53.14 ± 0.47 95.39 ± 0.15 -1.50 ± 0.01 1.08 ± 0.02 30.50 ± 0.15 -
H2 0 14.56 ± 0.00 3.76 ± 0.03 18.50 ± 0.00 32.74 ± 2.57 242.00 ± 1.00 5.31 ± 0.06 587.37 ± 13.23 83.35 ± 0.32 -0.73 ± 0.01 29.11 ± 0.03 0.00 Pure
H2MS5 5 14.43 ± 0.12 3.74 ± 0.01 17.67 ± 0.29 31.25 ± 0.00 232.00 ± 1.00 5.68 ± 0.03 556.20 ± 3.59 84.52 ± 0.11 -0.79 ± 0.02 28.47 ± 0.07 1.34 ± 0.22 Pure
H2MS10 10 14.36 ± 0.00 3.74 ± 0.01 17.00 ± 0.29 31.25 ± 0.00 223.00 ± 0.00 5.73 ± 0.04 517.58 ± 6.92 84.81 ± 0.18 -0.84 ± 0.01 27.63 ± 0.02 2.08 ± 0.18 Pure
HSMS20 20 14.23 ± 0.12 3.75 ± 0.00 15.33 ± 0.29 30.09 ± 2.01 204.00 ± 1.00 6.07 ± 0.04 482.20 ± 3.05 85.84 ± 0.58 -1.01 ± 0.02 24.97 ± 0.11 4.87 ± 0.38 Pure
HSMS30 30 14.16 ± 0.00 3.77 ± 0.00 13.83 ± 0.29 27.77 ± 0.00 186.80 ± 0.70 6.57 ± 0.08 418.29 ± 16.78 86.79 ± 0.17 -1.16 ± 0.02 22.16 ± 0.04 7.77 ± 0.18 Pure
H2MS40 40 14.09 ± 0.12 3.78 ± 0.01 12.33 ± 0.29 25.00 ± 0.00 168.87 ± 0.42 6.88 ± 0.02 373.47 ± 2.46 87.92 ± 0.26 -1.23 ± 0.01 20.38 ± 0.24 9.87 ± 0.32 Pure
H2MS50 50 14.03 ± 0.12 3.80 ± 0.01 10.83 ± 0.29 20.83 ± 0.00 148.53 ± 0.25 7.26 ± 0.20 320.47 ± 4.54 88.50 ± 0.21 -1.25 ± 0.01 16.65 ± 0.07 13.48 ± 0.08 Pure
MS 100 13.76 ± 0.00 5.42 ± 0.00 1.40 ± 0.10 0.00 ± 0.00 4.10 ± 0.00 10.53 ± 0.15 85.89 ± 0.39 95.31 ± 0.27 -1.40 ± 0.04 0.75 ± 0.04 30.78 ± 0.22 –

The values are expressed as mean ± standard deviation

European Food Research and Technology (2024) 250:2255–2272
European Food Research and Technology (2024) 250:2255–2272 2259

Table 4  Sugar profile of the Sample ID Ratio % F + G (g/100 g) F/G Maltose (g/100 g) Honey quality
samples
H1 0 72.93 ± 0.01 1.240 ± 0.01 1.87 ± 0.05 Pure
H1GFS5 5 70.60 ± 0.43 1.236 ± 0.01 3.15 ± 0.13 Pure
H1GFS10 10 68.37 ± 1.45 1.223 ± 0.01 4.20 ± 0.17 Pure
H1GFS20 20 65.02 ± 0.39 1.173 ± 0.05 5.94 ± 0.11 Adulterated
H1GFS30 30 61.12 ± 2.81 1.100 ± 0.03 7.33 ± 0.32 Adulterated
H1GFS40 40 56.47 ± 1.80 1.024 ± 0.02 9.01 ± 0.44 Adulterated
H1GFS50 50 51.31 ± 1.61 0.997 ± 0.01 10.17 ± 0.40 Adulterated
GFS 100 38.73 ± 0.60 0.759 ± 0.02 19.99 ± 0.59 –
H1 0 72.93 ± 0.01 1.240 ± 0.01 1.87 ± 0.05 Pure
H1MS5 5 70.34 ± 0.69 1.244 ± 0.01 3.58 ± 0.12 Pure
H1MS10 10 64.94 ± 2.29 1.232 ± 0.02 5.26 ± 0.19 Adulterated
H1MS20 20 58.75 ± 0.24 1.240 ± 0.02 8.99 ± 0.19 Adulterated
H1MS30 30 52.29 ± 1.29 1.165 ± 0.03 1328 ± 0.40 Adulterated
H1MS40 40 48.48 ± 0.46 1.110 ± 0.03 16.52 ± 0.16 Adulterated
H1MS50 50 38.90 ± 1.25 1.074 ± 0.01 19.93 ± 0.49 Adulterated
MS 100 2.19 ± 0.07 0.225 ± 0.00 38.19 ± 0.51 –
H2 0 73.06 ± 0.62 1.344 ± 0.03 3.00 ± 0.01 Pure
H2GFS5 5 71.44 ± 0.86 1.324 ± 0.00 3.93 ± 0.08 Pure
HSGFS10 10 69.28 ± 1.12 1.273 ± 0.01 4.92 ± 0.05 Pure
H2GFS20 20 66.58 ± 1.33 1.234 ± 0.04 6.92 ± 0.21 Adulterated
HSGFS30 30 63.00 ± 0.98 1.153 ± 0.02 8.82 ± 0.21 Adulterated
HSGFS40 40 60.19 ± 0.66 1.111 ± 0.02 10.86 ± 0.39 Adulterated
HSGFS50 50 56.93 ± 0.27 1.036 ± 0.03 12.58 ± 0.19 Adulterated
GFS 100 38.73 ± 0.60 0.759 ± 0.02 19.99 ± 0.59 –
H2 0 73.06 ± 0.62 1.344 ± 0.03 3.00 ± 0.01 Pure
H2MS5 5 69.93 ± 1.48 1.332 ± 0.02 5.35 ± 0.17 Adulterated
H2MS10 10 65.20 ± 0.77 1.349 ± 0.03 7.34 ± 0.18 Adulterated
HSMS20 20 59.17 ± 0.62 1.281 ± 0.03 10.78 ± 0.22 Adulterated
HSMS30 30 51.48 ± 2.23 1.256 ± 0.02 14.04 ± 0.54 Adulterated
HSMS40 40 45.27 ± 1.03 1.181 ± 0.04 17.34 ± 0.34 Adulterated
HSMS50 50 39.19 ± 0.53 1.204 ± 0.03 20.01 ± 0.45 Adulterated
MS 100 2.19 ± 0.07 0.225 ± 0.00 38.19 ± 0.51 –

The values are expressed as mean ± standard deviation

pH Electrical conductivity

10 g sample was dissolved in ­C02-free 75 ml pure water 20 g of sample dry matter was dissolved in 100 ml ultrapure
and pH measurement was performed after calibrating the water (18.2 MΩ.cm resistivity, Sartorius H2O-I-1-UV-T
pH meter (Orion 3-Star, Thermo Fisher Scientific Inc.) with Arium Comfort) and electrical conductivity of the sample
pH 4.00, 7.00 and 10.00 buffer solutions (Thermo Scientific solution was measured by a conductivity meter Consort
Inc.). C3010 (Consort bvba, Turnhout, Belgium) at 20 ℃. The
result was expressed as µS/cm.
Free acidity
Proline
Free acidity was performed by dissolving 10 g sample in
75 ml ­C02-free pure water and titrated with 0.05 M NaOH 5 g of sample was dissolved in pure water and then diluted
to pH 8.30 using a pH meter (Orion 3-Star, Thermo Fisher to a volume of 100 ml in a volumetric flask. 3% by vol-
Scientific Inc.). Free acidity was calculated as follows; ume ninhydrin (Sigma-Aldrich) in ethylene glycol mono-
Free acidity (mEq/kg) = 5 × volume of NaOH used in methyl ether (Fisher Chemical) and 40 mg/50 ml aqueous
titration (ml). L-proline (Merck) reference solution was prepared. 0.5 ml
2260 European Food Research and Technology (2024) 250:2255–2272

Table 5  δ13honey, δ13protein, and ­C4 sugar values of the samples

Sample ID Ratio % Honey δ13 C (‰) Protein δ13 C (‰) Protein–honey δ13 C (‰) C4 Sugar/(adultera- Honey quality
tion level) (%)

H1 0 − 25.80 ± 0.14 − 25.92 ± 0.08 − 0.12 ± 0.22 0.97 ± 1.02 Pure

H1GFS5 5 − 26.13 ± 0.07 − 26.51 ± 0.13 − 0.39 ± 0.07 3.64 ± 0.56 Pure
H1GFS10 10 − 25.34 ± 0.12 − 26.05 ± 0.12 − 0.71 ± 0.24 6.96 ± 2.30 Pure
H1GFS20 20 − 24.20 ± 0.15 − 25.72 ± 0.18 − 1.52 ± 0.03 15.37 ± 0.02 Adulterated
H1GFS30 30 − 22.95 ± 0.09 − 25.34 ± 0.74 − 2.39 ± 0.82 24.90 ± 6.76 Adulterated
H1GFS40 40 − 21.58 ± 0.12 − 25.19 ± 0.66 − 3.61 ± 0.78 35.25 ± 6.18 Adulterated
H1GFS50 50 − 20.44 ± 0.04 − 24.65 ± 0.18 − 4.21 ± 0.22 47.90 ± 1.52 Adulterated
GFS 100 − 15.86 ± 0.38 – – – –
H1 0 − 25.80 ± 0.14 − 25.92 ± 0.08 − 0.12 ± 0.22 0.97 ± 1.02 Pure
H1MS5 5 − 25.76 ± 0.09 − 26.16 ± 0.20 − 0.40 ± 0.30 3.97 ± 2.90 Pure
H1MS10 10 − 25.25 ± 0.11 − 26.02 ± 0.00 − 0.77 ± 0.11 7.79 ± 1.07 Adulterated
H1MS20 20 − 24.25 ± 0.01 − 25.75 ± 0.06 − 1.50 ± 0.05 15.64 ± 0.41 Adulterated
H1MS30 30 − 22.76 ± 0.13 − 25.15 ± 0.30 − 2.39 ± 0.43 26.47 ± 3.91 Adulterated
H1MS40 40 − 21.55 ± 0.32 − 25.23 ± 0.04 − 3.69 ± 0.36 40.52 ± 3.79 Adulterated
H1MS50 50 − 20.56 ± 0.24 − 24.92 ± 0.66 − 4.36 ± 0.90 49.42 ± 6.52 Adulterated
MS 100 − 15.86 ± 0.38 – – – –
H2 0 − 26.13 ± 0.23 − 26.08 ± 0.10 0.05 ± 0.34 1.45 ± 0.47 Pure
H2GFS5 5 − 25.67 ± 0.07 − 26.43 ± 0.06 − 0.75 ± 0.01 7.13 ± 0.10 Adulterated
HSGFS10 10 − 25.27 ± 0.07 − 26.20 ± 0.00 − 0.93 ± 0.06 8.95 ± 0.60 Adulterated
H2GFS20 20 − 24.27 ± 0.36 − 25.68 ± 0.04 − 1.41 ± 0.40 14.37 ± 4.03 Adulterated
HSGFS30 30 − 23.19 ± 0.04 − 25.38 ± 0.04 − 2.19 ± 0.00 23.01 ± 0.12 Adulterated
HSGFS40 40 − 21.84 ± 0.19 − 25.02 ± 0.53 − 3.18 ± 0.72 34.55 ± 5.86 Adulterated
HSGFS50 50 − 20.86 ± 0.07 − 25.04 ± 0.16 − 4.18 ± 0.09 45.57 ± 0.14 Adulterated
GFS 100 − 16.14 ± 0.87 – – – –
H2 0 − 26.13 ± 0.23 − 26.08 ± 0.10 0.05 ± 0,34 1.45 ± 0.47 Pure
H2MS5 5 − 25.78 ± 0.27 − 26.29 ± 0.09 − 0.51 ± 0,17 5.07 ± 1.71 Pure
H2MS10 10 − 25.22 ± 0.09 − 25.83 ± 0.17 − 0.62 ± 0,26 6.33 ± 2.54 Pure
HSMS20 20 − 24.22 ± 0.22 − 25.56 ± 0.15 − 1.34 ± 0,37 14.20 ± 3.68 Adulterated
HSMS30 30 − 23.32 ± 0.68 − 25.34 ± 0.81 − 2.01 ± 0,13 21.89 ± 0.50 Adulterated
H2MS40 40 − 21.14 ± 0.41 − 24.08 ± 0.48 − 2.94 ± 0,89 36.74 ± 9.00 Adulterated
H2MS50 50 − 20.42 ± 0.15 − 24.36 ± 1.00 − 3.95 ± 1,15 47.49 ± 8.18 Adulterated
MS 100 − 16.14 ± 0.87 – – – –

The values are expressed as mean ± standard deviation

sample solution, 0.5 ml blank solution (pure water), and a UV–Vis double beam spectrophotometer Jasco V-650 UV
0.5 ml prepared proline solution were added to each test (JASCO, Tokyo, Japan) at 510 nm in a 1 cm quartz cuvette
tube. Then 1 ml of formic acid (Sigma-Aldrich) and 1 ml (Hellma Analytics, Müllheim, Germany). Proline concentra-
of ninhydrin solution were added to each tube. The tube tion of samples was calculated using the following formula;
caps were screwed firmly and shaken at 400 rpm in a shaker Proline (mg/kg) = (Es/Ea) × (E1/E2) × 80, where Es is the
(IKA KS 4000 IC Control, Staufen, Germany) for 15 min at absorbance of the sample solution, Ea is the absorbance of
room temperature and then the tubes were subjected to heat the proline standard solution, E1 is the amount of proline
treatments. The tubes were first heated in a boiling bath for used for the standard solution (mg), E2 is the weight of
10 min and then immediately subjected to another heat treat- honey (gr), and 80 is the dilution factor [36].
ment in a 70 ℃ water bath for 15 min and to each tube, 5 ml
2-propanol (Carlo Erba) was added. After the heat treat-
ments, the tubes were left to cool down for 45 min at room
temperature and then proline content was determined using
European Food Research and Technology (2024) 250:2255–2272 2261

Diastase number (Agilent, 4,6 × 250 mm 5 μm). Analysis was performed in

isocratic mode (80:20 acetonitrile (HPLC grade, Sigma-
The diastase activity was calculated as diastase number Aldrich)/water at 1.3 ml/min flow rate) at 30 ℃ column and
(DN) in Gothe units. DN is equal to % the amount of 1% detector temperature. Seven-point calibration curves were
starch solution (ml) that the diastase enzyme in 1 g of honey prepared for glucose (D-glucose anhydrous, Fluka, 300 ppm-
can completely hydrolyze in 1 h. The test was carried out 25000 ppm), fructose (D(-)-Fruktoz, Sigma-Aldrich,
according to the Turkish Standards Institution [37]. 300 ppm-25,000 ppm), sucrose (Fluka, 10 ppm-2000 ppm),
and maltose (D-( +)-maltose monohydrate, Fluka, 300 ppm-
Color 25000 ppm) sugar standards. An example of the individual
sugar profile HPLC chromatogram is provided in the sup-
To completely dissolve sugar crystals, the sample was heated plementary document (see Fig. S1).
to 50 ℃ in a water bath and then transferred into a spectro-
photometer cuvette (Biosigma SpA, Italy). Color measure- C4 sugars by IR‑MS
ment was conducted using a chroma meter (Konica Minolta
CR5, Japan) and L, a, and b values were obtained at 25 ℃. For the extraction of protein from honey, 12 g of the sam-
L, a, and b parameters were used to calculate the ΔE value ple was put into a 50 ml centrifuge tube, diluted with 4 ml
which was calculated as follows; ultrapure water, and vortexed until the sample fully dis-
1∕2 solved. Then 2 ml of 10% tungstic acid sodium salt solu-
ΔE = [(L − L0)2 + (a − a0)2 + (b − b0)2 ] tion and 2 ml of 0.335 M sulfuric acid was added to the
sample solution, the tube was then placed in a water bath at
80 ℃ for 30 min. Following that, 1 ml of 10% tungstic acid
Hydroxyl methyl furfural (HMF) by HPLC–DAD sodium salt solution and 1 ml of 0.335 M sulfuric acid were
added into the tube and this step was repeated three times.
10 g of sample was diluted to 50 ml with ultrapure water After this heat treatment, 25 ml ultrapure water was added
(18.2 MΩ.cm resistivity, Sartorius H2O-I-1-UV-T Ari- to the tube and the solution was centrifuged at 1500 rpm for
uim Comfort) and filtered through 0.45 μm Minisart filter 5 min at room temperature. After centrifugation, the super-
(Sartorius Stedim Biotech GmbG, Goettingen, Germany). natant was removed and the tube was filled to the mark with
A stock solution of HMF (100 ppm) was prepared with a ultrapure water. This centrifugation was repeated four times
standard HMF (J&K, Haihang Industry Co., Ltd.) and then until the supernatant was clear. The supernatant was dis-
using the stock solution, six-point calibration curve of HMF carded, the pellet was transferred to a watch glass, and left to
in the range of 0.1 ppm–10 ppm was prepared. HMF con- dry in an oven at 70 ℃ for 45 min. Between 150 and 200 µg
tent was determined using a HPLC instrument (1260 Infin- dried protein, honey and syrup samples were weighed in
ity II, Agilent Technologies) equipped with a diode array each tin capsule and then were tightly closed. Packed sam-
detector (1260 Infinity II WR) and C18 column (ACE 5, ples were then introduced into the autosampler of the EA
250 mm × 4.6 mm, 5 μm). Analysis was performed in iso- IR-MS system (DELTA V Plus, Thermo Fisher Scientific).
cratic mode (90:10 water: methanol at 1 ml/min flow rate) The degree of adulteration of sample was calculated accord-
operated at 285 nm, at 30 ℃ column temperature. ing to the AOAC method 998.12 [38] by the equation given
below;
[ ]
(𝛿‰ protein − 𝛿‰ honey)
Sugar profile by HPLC‑RID % Adulteration = x100
(𝛿‰ protein − 𝛿‰ syrup)

5 g of sample was weighed into a beaker and dissolved in

ultrapure water (18.2 MΩ.cm resistivity, Sartorius H2O-
I-1-UV-T Arium Comfort). 25 ml methanol (Chromasolv Results and discussion
grade, Sigma-Aldrich) was pipetted into a 100 ml volumetric
flask and then filled to the mark with sample solution and Physico-chemical parameters (moisture content, pH, free
ultrapure water. The prepared methanol sample solution was acidity, diastase number, electrical conductivity, HMF, pro-
filtered through a 0.45 μm filter (Sartorius Stedim Biotech line, color) of blossom honey samples (H1 and H2), adul-
GmbG, Goettingen, Germany). Determination of sugars terants (MS and GFS syrups), and adulterated honey sam-
was carried out using an HPLC instrument (1260 Infinity ples (H1GFS, H1MS, H2GFS, and H2MS) are presented
II, Agilent Technologies) equipped with a refractive index in Table 3. HPLC-RID sugar profile and IR-MS C4 sugar
detector (RID, 1260 Infinity II) and Zorbax NH2 column analysis are given in Tables 4 and 5, respectively.
2262 European Food Research and Technology (2024) 250:2255–2272

Moisture content inhibited [14]. The early pH values of H1 and H2 honey

samples and GFS and MS syrups were observed as 3.70,
In terms of honey quality, moisture content is one of the 3.76, 5.42, and 5.19 respectively (see Table 3). In general,
most important parameters that affects the physico-chemical the pH values of the adulterated samples were increased with
properties of honey and will help to determine the storage the increase in syrup addition levels. Since MS syrup has a
conditions and shelf life of the product. Moisture content higher pH value than GFS syrup, the increase in pH levels
in honey above 20% can trigger yeast growth which sub- of adulterated samples in the samples prepared with MS
sequently causes the fermentation process [11]. The initial syrup was higher than in samples prepared with GFS syrup.
moisture content of H1 and H2 honey samples and GFS Among the adulterated samples, the highest pH value was
and MS adulterants was found as 14.96%, 14.56%, 17.16%, examined in the B2MS50 sample as 3.80. Considering the
and 13.76%, respectively (see Table 3). There were linear pH values of the syrup and honey samples, changes in the
downward with the addition of GFS syrup and linear upward pH values of adulterated samples were found lower than
trends with the addition of MS syrup (see Fig. 1). Since expected (Figure S2). This could be due to the buffering
both sugar syrups have lower than 20% moisture content, all property of the honey matrix that keeps the pH values of the
adulterated samples ranged below 20% which do not exceed adulterated samples at lower levels.
the maximum limit of moisture specified in the Turkish Food (Transferred to Supplementary material as Fig. S2) Simi-
Codex Communiqué on honey [10]. lar results were reported in a previous study [14], in which
Among the adulterated samples, the highest moisture the addition of HFCS at different ratios (10%, 25%, 50% and,
content was seen in the H1GFS50 sample at the amount of 75%) to pure honey samples increased honey’s pH value. Ini-
15.96%. tially honey and HFCS had 3.10 and 4.70 pH values, respec-
In a study, different honey samples were adulterated with tively. An increase in the addition level of HFCS increased
sucrose beet syrup at the ratio of 10–50% and the moisture pure honey’s pH rate. In the adulterated honey samples, the
content of honeys exceeded the 20% limit at 40% and 50% pH rates ranged between 3.30 and 3.98.
adulteration levels [12]. In another study [13], it was deter-
mined that the moisture content of honey adulterated with Free acidity
palm sugar syrup in the range of 5–30% decreased from
19.7% to 21.6% with the increase of the added syrup level. The main source of free acidity is the presence of organic
By examining the data obtained from this study and current acids. Although organic acids are around 0.5% of the total
literature, it can be said that it is not always possible to detect honey composition, they have important roles in the organo-
adulterated honey according to their moisture content. leptic, physical, and chemical properties of honey [15]. Free
acidity value is also seen as a fermentation indicator. Bad
pH storage conditions, exposure to direct heat, microbial con-
tamination, and/or components that are decomposed by the
The value of pH is another parameter that has an impact naturally occurring osmophilic yeasts in honey cause an
on the characterization of honey. In the presence of low increase in the free acidity value [16, 17]. Free acidity of
pH, microbial growth as well as microbial reproduction is H1 and H2 honey samples was measured as 27.00 mEq/kg

Fig. 1  Variation in moisture

content of the samples at differ- 0.993 0.931
ent syrup ratios (the red dashed .995 0.912
lines represent the permitted 20 20
maximum level)
18 18

16 16

14 14

12 12

10 10
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100
European Food Research and Technology (2024) 250:2255–2272 2263

Fig. 2  Variation in free acidity

of the samples at different syrup
0.996 0.995 0.992 0.996
ratios (the red dashed lines rep-
resent the permitted maximum 50 50
level)
40 40

30 30

20 20

10 10

0 0
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100

and 18.50 mEq/kg, respectively, while the value for GFS 17.89. DN number values of all adulterated samples were
and MS syrup was found as 1.63 mEq/kg and 1.40 mEq/kg, found over 8.00 (see Fig. 3) which is in the acceptable range
respectively (see Table 3). It was determined that there were stated in EC Council Directive [9] and Turkish Food Codex
high negative correlations between the free acidity values Communiqué on honey [10].
and the amount of syrup added to the samples. As the syrup Pure honey samples were adulterated with sucrose syrup
addition level was increased, the free acidity values of the at the range of 10–50%. Due to the absence of diastase activ-
adulterated samples were decreased linearly (Fig. 2). Free ity in sucrose syrup, the average DN of the pure honey sam-
acidity values of samples ranged between 24.17 mEq/kg and ples gradually decreased from 14.60 to 7.50 with increas-
17.17 mEq/kg depending on adulteration levels. ing adulteration levels [12]. Czipa et al. in their study [20]
In a study [18], glucose, hydrolyzed inulin syrup, malt directly added a different type of sugar syrups (glucose,
wort, and inverted sugar were used for the adulteration of invert and fructose-glucose syrup) at a ratio of 30% and 40%
different authentic honeys at ratio of 5%, 10%, 20%, 30%, to pure acacia honey samples. They reported that the aver-
40%, and 50% respectively. It was observed that glucose, age DN of samples was 28.80 and depending on the type of
hydrolyzed inulin syrup, malt wort, and inverted sugar syr- sugar syrup added, the value decreased down to 15.40. In
ups increased the amount of free acidity, although fructose a study conducted by Ozcan et al. [21], the diastase activi-
did not alter the free acidity values of the samples. Average ties of honey obtained from bees fed with sucrose syrup
free acidity of authentic honey went up from 19.44 mEq/kg and inverted syrup were compared with honey obtained
to 162.88 mEq/kg with the addition of 50% sugar syrups. from bees not fed with sugar. The highest diastase value
According to the Turkish Food Codex Communiqué on was found in pure honey with 10.90 and the lowest diastase
honey [10] and Council Directive (2001), honey should not value was determined in honey obtained from bees fed with
have more than 50 mEq/kg free acidity; however, there is no invert syrup. The diastase activity of honey produced by
minimum tolerance level for free acidity. In this study, since bees fed with sucrose syrup was found to be 8.30. It can be
all the adulterated samples remained lower than 50 mEq/kg, seen from our study and the literature that direct or indirect
no adulterations were detected among the samples (Fig. 2). addition of sugar syrups decreases the diastase activity of
honey but the DN value of the adulterated samples mostly
Diastase number (DN) remains within the safe limit which is the amount of 8.00
DN set by Turkish Honey Communiqué [10] and Council
Diastase is considered an indicator of the freshness and Directive [9] as a minimum requirement.
purity of honey [19]. The initial DN of the H1 and H2 honey
samples was determined as 28.93 and 32.74 while diastase Electrical conductivity
activity was not found in both GFS and MS syrups. There
were negative correlations between the amount of syrup The amount of electrical conductivity (EC) varies directly
added and the diastase numbers of the adulterated samples. with the presence of mineral substances, organic acids, and
DN of the adulterated honey samples was generally gradu- other organic compounds [22]. The EC values of H1 and H2
ally decreased with the increase of syrups added. The DN honey were determined as 307.00 μS/cm and 242.00 μS/cm,
of the adulterated honey samples ranged between 31.25 and respectively, while the EC values of GFS and MS syrups
2264 European Food Research and Technology (2024) 250:2255–2272

Fig. 3  Variation in diastase

number of the samples at differ-
ent syrup ratios (the blue dashed 0 0.917 0 0.953
lines represent the permitted
minimum level)

Fig. 4  Variation in electrical

conductivity of the samples 0 0.982
0 0.990
at different syrup ratios (the
red dashed lines represent the 800 800
permitted maximum level) 700 700
600 600
500 500
400 400
300 300
200 200
100 100
0 0
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100

were found to be 5.20 μS/cm and 3.00 μS/cm, respectively cm in B1GFS5 and B2MS50 samples, respectively (see
(see Table 3). In the measurements of adulterated sam- Table 3). These results show that according to the regula-
ples, high negative correlations were detected between the tions, the addition of sugar syrups to blossom honey affected
added syrup level and the EC of adulterated samples. It positively the electrical conductivity value so there should
was determined that the amount of electrical conductivity also be a minimum requirement for the electrical conductiv-
decreased proportionally as the amount of added sugar syr- ity requirement of blossom honey in the regulation.
ups increased (see Fig. 4).
Oroian et al. [23] reported that EC values of different HMF
types of honey (acacia, tilia, and polyfloral) were altered
by adulteration with fructose and hydrolyzed inulin syrups. HMF is an indicator of honey freshness [24]. It is known
While the addition of fructose syrup decreased, hydrolyzed that sugar syrups added to honey generally increase the
inulin increased the EC value of the samples as the adultera- HMF content due to the higher presence of HMF in sugar
tion rate increased. EC values of the adulterated samples syrups [25]. Furthermore, for the added sugars to disperse
were found to be between 24.30 and 2920 μS/cm. According homogeneously in the honey–syrup mixture and gain a
to Turkish Honey Communiqué regulation [10], EC value uniform appearance, the mixture is heated after adding the
for blossom honey should be less than 800.00 μS/cm. In our syrup, and this process causes an increase in the amount
study, blossom honey samples and all the adulterated honey of HMF. While the HMF content of H1 and H2 honey was
samples showed complete conformity to this regulation. The determined as 7.12 mg/kg and 5.21 mg/kg, respectively,
highest and the lowest conductivity values in adulterated in GFS and MS syrups, HMF contents were found as
samples were determined as 295.00 μS /cm and 149.87 μS/ 16.96 mg/kg and 10.53 mg/kg, respectively (see Table 3).
European Food Research and Technology (2024) 250:2255–2272 2265

Fig. 5  Variation in HMF con-

tent of the samples at different
.925 .974
syrup ratios (the red dashed
lines represent the permitted
maximum level)

As the syrup addition level increased in the adulterated of HMF, adulterated honeys mostly did not exceed the limit
samples, the amount of HMF increased linearly showing of 40 mg/kg set by the Turkish Food Codex Communiqué
high positive correlations (see Fig. 5). HMF content of on honey [10].
adulterated honey samples was ranged between 5.68 mg/
kg and 11.94 mg/kg. Proline content
In a study conducted by Craciun et al. [26], honey sam-
ples were adulterated by adding three different sugar syr- Salivary glands of honey bees and plants are the main
ups directly to authentic honey and indirectly feeding the sources of amino acids of honey counted as an indicator
bees with sugar syrups. While the average HMF content in determining honey fraud whether it has been imitated or
of authentic honeys was 1.21 mg/kg, the average HMF of adulterated. Proline is the dominant amino acid in honey and
directly syrup-added honeys was found to be 21.20 mg/ it is seen as an indicator of protein amount in honey since it
kg. Furthermore, the average HMF value of adulterated constitutes 50–85% of the total amino acid content [27, 28].
honey obtained from bees fed with sugar syrups was found In H1 and H2 honey samples, proline contents were found
as 29.90 mg/kg. In another study, monofloral acacia, tilia, as 965.54 mg/kg and 587.37 mg/kg, respectively. Proline
and sunflower honeys were adulterated with maple, inverted values of GFS and MS syrups were calculated as 53.13 mg/
sugar, agave, rice and corn syrup in the concentration of 5%, kg and 85.88 mg/kg, respectively (see Table 3). High linear
10%, and 20%. Initially, honey samples had an average of negative correlations were determined between the addition
3.50 mg/kg HMF content with the addition of sugar syrups rate of syrups and the proline content of adulterated samples
and average HMF values of the samples increased gradually (see Fig. 6).
in the range of 10.10 to 35.10 mg/kg [5]. Although sugar The highest and the lowest amounts of proline were found
syrups used for the adulteration of honeys had higher content in H1MS5 and H2GFS50 at 902.53 mg/kg and 314.96 mg/

Fig. 6  Variation in proline con-

tent of the samples at different
syrup ratios (the blue dashed 0 0.992 0.996 0.997
lines represent the permitted 1.000 1.000
minimum level) 900 900
800 800
700 700
600 600
500 500
400 400
300 300
200 200
100 100
0 0
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100
2266 European Food Research and Technology (2024) 250:2255–2272

kg, respectively. In the study conducted by Kropf et al. [20], with syrup added increased and b value decreased propor-
a pure honey sample was adulterated with fructose, glucose, tionally with the syrup addition rate. Yılmaz et al. [31], in
and two different inverted sugar syrups at the rates of 30% their study, detected the changes in L, a, and b values by
and 40%. While the proline value of pure honey was deter- adulterating a honey sample with fructose and sucrose syr-
mined as 284.00 mg/kg, it was also determined that the pro- ups in the range of 5–50%. With the increase in the syrup
line values of the syrup-added honey samples decreased and ratio added to the honey sample, an increase in L value and
the proline values of the samples ranged from 179.00 mg/ a decrease in a and b values were determined. While the
kg to 274.00 mg/kg. In another study, proline values of highest L value was detected in the sample with 50% sucrose
honey obtained from bees fed with sucrose syrup at differ- syrup added, the lowest a and b values were determined in
ent rates were compared. Proline amounts of 416.40 mg/ the samples including 50% sucrose syrup and 50% fructose
kg, 501.60 mg/kg, and 630.00 mg/kg were determined in syrup, respectively.
honey obtained from bees fed with sugar syrup continuously,
fed only with sugar syrup in spring, and not fed with sugar
syrup, respectively [29]. Sugar profile by HPLC

Color To prevent imitation and adulteration, there are certain

criteria set by national and international standards for the
Color variability in honey depends on the nectar of plants sugar content of honey. According to the Turkish Food
and the plant origin because honeybees directly collect Codex Communiqué on honey [10], maltose and sucrose
nectar and pollen from plants. It plays an important role can be found at a maximum of 4 (g/100 g) and 5(g/100 g)
in determining the market price of honey as it is one of the respectively, and the F + G value should be at least 60%, and
most important physical parameters affecting the preferences the fructose/glucose ratio should be in the range of 0.9–1.4.
of consumers in many countries [30]. As a result of the color Honey that does not meet these criteria is considered as
test, the L, a, and b values were obtained. Color differences, fraudulent. In the adulterated honey samples, fructose + glu-
ΔE, were generated from the L, a, and b values of the sam- cose (g/100 g) values and F/G ratios were decreased gradu-
ples. Raising the percentage of adulterants, GFS and MS, in ally (Figs. 7 and 8, respectively), while maltose (g/100 g)
the adulterated samples caused a gradually increase in the values progressively increased with increasing additions of
L and ΔE values (see Figures S3 and S4) while gradually GFS and MS syrups (Fig. 9).
causing decrease in a and b values (see Figures S5 and S6). Except for H1MS50 and H2MS50, all the adulterated
The highest L and ΔE values were detected in the B2GFS50 samples contain more than 60% of F + G. Furthermore F/G
and B1MS50 samples. The lowest a and b values were found ratio of all adulterated samples ranged between 0.9 and 1.4.
in the samples of B2GFS50 and B2MS40, respectively (see Sucrose sugar was not found in H1 and H2 honey samples
Table 3). and in both GFS and MS syrups. Results of F + G (g/100 g),
In a study carried out by Ribeiro et al. [14], honey sam- F/G ratio, and maltose (g/100 g) are exhibited in Table 4.
ples were adulterated with high-fructose corn syrup at a rate These results indicate that adulterations are detected at the
of 10–75%. It was found that, while the L value of honeys level of 5%—50% depending on the sugar syrup and honey

Fig. 7  Variation in F + G value

of the samples at different syrup
ratios (the blue dashed lines 0 0.996 0 0.999
represent the permitted mini- 80
mum level)
60

40

20

0
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100
European Food Research and Technology (2024) 250:2255–2272 2267

Fig. 8  Variation in F/G value of

the samples at different syrup
ratios (the red dashed lines 0 0.859 0 0 .845
represent permitted maximum 2 2
level; the blue dashed lines rep-
resent the permitted minimum
level) 1.4 1.4

1 1
0.9 0.9

0 0
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100

Fig. 9  Variation in maltose

value of the samples at different
syrup ratios (the red dashed .999 .998
lines represent the permitted 40 40
maximum level) 36 36
32 32
28 28
24 24
20 20
16 16
12 12
8 8
4 4
0 0
0 5 10 20 30 40 50 100 0 5 10 20 30 40 50 100

Fig. 10  Variation in ­C4 sugar

value of the samples at different
syrup ratios (the red dashed .989 .974
lines represent the permitted 50 50
maximum level)
40 40

30 30

20 20

10 10
7 7
0 0
0 5 10 20 30 40 50 0 5 10 20 30 40 50

type. In the samples of H1GFS, H1MS, H2GFS, and H2MS, In a study [32], honeydew honey was adulterated with
adulteration was detected at ratio of starting from 20%, 10%, glucose, fructose, inverted sugar, hydrolyzed inulin syrup,
20%, and 5% respectively. and malt wort at the rate of 5–50%. According to the EC
Council Directive [9], adulterations could be detected in
2268 European Food Research and Technology (2024) 250:2255–2272

samples which were adulterated with glucose, fructose, C4 sugars by IR‑MS

and malt-worth additions at the rate of between 20–50%,
5–50%, and 20–50%, respectively. Adulterations were dis- According to the Turkish Food Codex Communiqué on
covered by considering the F/G ratio and maltose ratio of the honey [10], the ratio of C4 should not be greater than 7%,
adulterated samples. The F/G ratio of adulterated samples δ13 protein—δ13 honey (‰) value should not be greater
ranged between 1.39–3.68 and 1.12–0.37, respectively. Fur- than ‰1, and the honey δ13 C value should be—23 (‰) or
thermore, the maltose value of adulterated with malt worth more negative (See Table 2). The δ13 C honey (‰) values
ranged between 3.0 and 24.92 (g/100 g). In a similar study of H1 and H2 pure honey samples were found as − 25.80
carried out by Tosun [33], three different blossom honey and − 26.13, respectively, while the values for GFS and MS
samples were adulterated with glucose, sucrose, and HFCS syrups were detected as -15.86 and − 16.14, respectively. C4
in the range of between 10% and 50%. According to the sugar adulteration levels (%) of the samples were increased
author’s results, the F + G ratio of three honey samples went with increasing ratio of sugar syrups in the samples (See
below 0.9 with the addition of glucose syrup at a rate of 40% Fig. 10).
and 50%. The F/G ratio of samples ranged between 0.54 As can be seen from Table 5, adulterations were detected
and 0.77. The F + G value of adulterated samples was found at the level ranged between 5 and 50% depending on honey
within safe limits which were more than 60 g/100 g. and sugar syrup type using EA-IRMS.

Table 6  Statistical analysis of the test results for samples prepared with H1 honey (H1-GFS, H1-MS)
Dependent variable Independent variable Sequential sum of Computed F P value
squares

Moisture Syrup type 16.803 2016.40 0.000

Syrup ratio 1.330 22.80 0.000
pH Syrup type 5.2 × ­10−5 0.13 0.721
Syrup ratio 13.017 4648.89 0.000
Free acidity Syrup type 0.677 4.94 0.033
Syrup ratio 2694.48 2812.24 0.000
Diastase number Syrup type 3.876 1.63 0.211
Syrup ratio 4065.078 244.30 0.000
Electrical conductivity Syrup type 14.192 9218165 0.000
Syrup ratio 400068,40 9218165 0.000
HMF Syrup type 62.609 1754.57 0.000
Syrup ratio 183.86 736.079 0.000
Proline Syrup type 7762.026 67.137 0.000
Syrup ratio 3586929.00 4432.15 0.000
IR-MS Syrup type 13.748 1.095 0.313
C4 Sugar Syrup ratio 7911.784 104.989 0.000
HPLC
F+G Syrup type 1069.552 663.171 0.000
Syrup ratio 12269.890 1086.841 0.000
F/G Syrup type 0.09 19.651 0.000
Syrup ratio 2.645 843.508 0.000
Maltose Syrup type 395.813 3883.553 0.000
Syrup ratio 3235.535 462.219 0.000
Color
L Syrup type 4.296 103.472 0.000
Syrup ratio 1846.864 5354.664 0.000
a Syrup type 0.72 1.655 0.207
Syrup ratio 156.663 513.951 0.000
b Syrup type 3.040 48.816 0.000
Syrup ratio 10410.262 23880.058 0.000
ΔE Syrup type 0.272 3.522 0.70
Syrup ratio 12285.965 22769.356 0.000
European Food Research and Technology (2024) 250:2255–2272 2269

Table 7  Statistical analysis Dependent variable Independent variable Sequential sum Computed F P value
of the test results for samples of squares
prepared with H2 honey
(H2-GFS, H2-MS) Moisture Syrup type 15.641 240.873 0.000
Syrup ratio 4.473 2085.444 0.000
pH Syrup type 0.010 21.143 0.000
Syrup ratio 12.441 3860.03 0.000
Free acidity Syrup type 0.935 14.622 0.001
Syrup ratio 1272.16 2841.492 0.000
Diastase number Syrup type 3.625 1.953 0.172
Syrup ratio 4819.591 371.059 0.000
Electrical conductivity Syrup type 14.410 28.784 0.000
Syrup ratio 245216,31 69974.33 0.000
HMF Syrup type 55.966 4551.59 0.000
Syrup ratio 306.984 3566.64 0.000
Proline Syrup type 1997.062 21.780 0.000
Syrup ratio 1145691 1784.953 0.000
IR-MS Syrup type 0.439 0.031 0.863
Syrup ratio 6688.667 70.090 0.000
HPLC F+G Syrup type 1639.523 1649.360 0.000
Syrup ratio 12136.010 1744.119 0.000
F/G Syrup type 0.01 0.857 0.361
Syrup ratio 3.271 593.311 0.000
Maltose Syrup type 380.273 3793.518 0.000
Syrup ratio 2962.931 4222.508 0.000
Color L Syrup type 1.343 10.720 0.003
Syrup ratio 605.160 689.863 0.000
a Syrup type 0.037 40.294 0.000
Syrup ratio 3.076 480.424 0.000
b Syrup type 0.111 5.822 0.022
Syrup ratio 3614.059 27039.955 0.000
ΔE Syrup type 0.053 0.615 0.439
Syrup ratio 4716.178 6969.081 0.000

Table 8  Results of the PCA Sample H1 H2

analysis using data obtained
from moisture, pH, free acidity, Principle com- Eigenvalues Eigenvalues
diastase number, electrical ponent
conductivity, HMF, and proline Variability (%) Cumulative (%) Variability (%) Cumulative (%)
analyses of the samples
PC1 80.230 80.230 83.898 83.898
PC2 16.937 97.167 11.924 95.821
PC3 1.715 98.882 3.518 99.339
PC4 0.807 99.689 0.386 99.725
PC5 0.158 99.847 0.129 99.854
PC6 0.059 99.906 0.068 99.922
PC7 0.051 99.958 0.049 99.971
PC8 0.027 99.984 0.016 99.987
PC9 0.014 99.999 0.010 99.998
PC10 0.001 100.000 0.002 100.000

The bold values belong to the F1 + F2 axes

2270 European Food Research and Technology (2024) 250:2255–2272

Fig. 11  Principal component

scores of the authentic honey
samples and honey samples
adulterated with GFS and MS
syrup

In a study [34], honey samples were adulterated with Principle component analysis (PCA)
high-fructose corn syrup, glucose syrup, and sucrose syrup
by the addition level of 10%, 20%, 30%, 40%, and 50%. In According to results obtained from moisture, pH, free acid-
this study, although adulterations could not be detected in ity, diastase number, electrical conductivity, HMF, and pro-
samples adulterated with glucose syrup and sucrose syrup, line content analyses, we were not able to determine adul-
adulterations made with high-fructose corn syrup were teration by these routine laboratory methods. Because, PCA
detected at the rate of 20%, 30%, 40%, 50%, as 14.30%, software (XLSTAT, 2021, Addinsoft, New York, NY) was
32.80%, and 41.65%, respectively. In a similar study, honey used to detect adulteration based on the physico-chemical
samples adulterated with high-fructose corn syrup at rates of parameters of pure honey and adulterated honey samples.
20%, 60%, 90% were analyzed, revealing adulteration levels By utilizing PCA, adulterations were detected at the range
of 11.20%, 30.60%, and 48.20%, respectively [35]. of 5–50% levels. It can be seen from the data obtained from
PCA results, as shown in Table 8 and Fig. 11, that two dif-
ferent PC values F1 and F2 were enough to classify the blos-
Statistical analyses som honey samples and the adulterated honey samples.
The resulting data matrix included 18 variables which are
SPSS analysis moisture, pH, free acidity, diastase number, electrical con-
ductivity, HMF, color (L, a, b, and ΔE), and proline values
IBM SPSS version 22 was used to generate statistical data of the samples. The first two PCAs scores, F1 + F2 axes were
through general linear model (GLM) at significance level (p able to explain 95.82% and 97.17% total variance of the sam-
value) of 0.05. Syrup type and syrup ratio were established ples prepared with H1 and H2 honey samples, respectively.
as independent variables whereas moisture, pH, free acid- These high percentages of total variances indicate the suc-
ity, diastase number, electrical conductivity, HMF, proline, cess of the PCA for the classification of the honey samples.
C4 sugar, F + G, F/G, maltose, L, a, b, and ΔE values were It can be observed from Fig. 11, the PCA score plot for H1
chosen as dependent variables. All the experimental results and H2 samples, both pure honey samples were settled at the
are presented in Tables 6 and 7. In Table 6, the results of same place on the left side of the plot near to origin point.
samples prepared with H1 honey, and in Table 7, results of Furthermore, both H1 and H2 samples adulterated with GFS
samples prepared with H2 are listed. For the results of sam- and MS syrups at the range of 5%, 10%, 20%, 30%, 40%,
ples prepared with H1 and H2 honey samples, independent and 50% were also clustered between each other based on
variables, syrup type and syrup ratio, had statistically signifi- adulteration levels in the same quadrant.
cant effects on moisture, free acidity, electrical conductivity,
HMF, proline, F + G, F/G, maltose, L and b values, while
syrup type was not significant for pH diastase number and Conclusion
C4 sugar, while syrup type was not significant (p > 0.05)
for diastase number, C4 sugar, ΔE values of the samples. According to the results of the experimental tests, several
Furthermore, syrup type did not significantly impact on pH conclusions can be drawn regarding the adulterated honey
and value of the samples prepared with H1 honey. samples. First, the free acidity, proline, diastase number,
electrical conductivity, a and b values of the adulterated
European Food Research and Technology (2024) 250:2255–2272 2271

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Authors and Affiliations

Ramazan Gün1,2 · Mehmet Murat Karaoğlu3

2
* Ramazan Gün Central Laboratory Application and Research Center, Bingöl
[email protected] University, 12000 Bingöl, Turkey
3
1 Department of Food Engineering, Faculty of Agriculture,
Department of Nutrition and Dietetics, Faculty of Health
Atatürk University, 12000 Erzurum, Turkey
Sciences, Bingöl University, 12000 Bingöl, Turkey